The [4fe4s] Cluster Of Yeast Dna Polymerase E Is Redox Active And Can Undergo Dna-Mediated Signaling

Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase epsilon (Pol epsilon) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2(CORE,) are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2(CORE)exo(-), was used to limit DNA degradation. While no redox signal is apparent for Pol2(CORE)exo(-) on DNA-modified electrodes, a large cathodic signal centered at -140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2(CORE)exo(-), which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S](2+), to oxidized Pol2(CORE)exo(-) bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIII(Y82A), a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2(CORE)exo(-). We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2(CORE)exo(-) via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol epsilon.